Summary: During FY17, we made progress on several subprojects: 1) Herpesvirus Assembly. Over the past 25 years, we have studied many aspects of herpesvirus assembly: most notably, how the elaborate capsid is formed and how it matures. Over the past year we continued this line of investigation with the focus shifting to nuclear egress. The HSV-1 capsid assembles in the nucleus, then transfers into the cytoplasm for subsequent steps on the pathway. The Primary Enveloped Virion is a transient particle formed in the perinuclear space as the DNA-filled capsid traverses the nuclear envelope. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a Primary Enveloped Virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. To obtain enough PEVs to support structural analysis, we isolated them from US3-null-infected cells (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space). This material was studied by cryo-EM and cryo-ET. We found that PEVs differ from mature extracellular virions (MEVs) in several fundamental respects. PEVs have very few glycoprotein spikes whereas MEVs are densely coated with them. PEVs are 20% smaller than MEVs and there is little space between the capsid and the NEC layer whereas in a MEV, this space is more capacious and is occupied by the tegument, a loosely ordered ensemble of viral proteins. The NEC has been proposed to be organized as hexamers in honeycomb arrays but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, the NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions. 2) Packing of DNA in bacteriophage T4, Part I. In previous work, our cryo-EM studies of bacteriophage T7 provided strong evidence in support of the concentric spool model whereby the DNA is coiled around the portal axis in nested shells (Cerritelli et al., Cell 91, 271-290 1997). In the present undertaking, we investigated whether a similar arrangement pertains in T4 which has a 4-fold larger genome (172 kbp), a prolate capsid, and lacks the cylindrical protein core of T7. Prolate heads were imaged in side-views and axial views, defined as such relative to the axis running through the portal vertex. We also imaged isometric heads in side-view, axial view, and along the axis of a non-portal vertex; and giant heads in side-view. Partially filled heads gave clues as to intermediate states on the packaging pathway. To observe the effect of duplex DNA stiffness on its packing, a tagged nuclease encapsidated during prohead assembly was activated by adding calcium. This treatment digested the genome to segments of 1 kbp, enhancing the crystallinity of packing. We conclude that in isometric T4 heads, the DNA is spooled around the portal axis, essentially as in T7; in prolate heads, the spool has elliptical gyres and the spooling axis is rotated by 60o relative to the portal axis; in giant heads, this angle increases to 90o. The innermost regions of DNA-filled heads are less ordered. We infer that the conformations of encapsidated genomes represent an energy-minimized compromise between electrostatic repulsion effects involving DNA duplexes and the strain associated with bending the DNA. It is the latter effect that reorients the spool in axially extended capsids. 3) Bubblegram Imaging: Packing of DNA in bacteriophage T4, Part II. While many approaches can be used to label proteins that are exposed on the surface of a macromolecular complex, few are applicable to proteins that are buried inside. We have been developing the use of radiation damage in vitrified specimens for that purpose. Sustained exposure to the electron beam elicits the formation of bubbles of hydrogen gas in proteins. The locations of the bubbles can be determined in the irradiated complex and related to native structure by comparing them with 3D density maps calculated from previously recorded low-dose images of the same specimen, then undamaged. We call this approach bubblegram imaging. Our first success (Wu et al., Science 335, 182 2012) was with PhiKZ, a large and complex bacterial virus that infects Pseudomonas aeruginosa. This was followed in FY14 with characterization of the internal structure of bacteriophage T7, viewed as a partially defined model system. In FY16 we completed a bubblegram analysis of herpes simplex virus capsids in which we detected a protein putatively the viral protease - underlying the capsid vertices (see above) and one of bacteriophage P22 in which we localized its ejection proteins. In FY17, we turned to bacteriophage T4, seen as a model system for large and structurally complex virions. Up to this point, we had detected internal proteins in several bacteriophages - in each case, in the form of a large, ordered complex associated with the inner surface of the portal vertex. PhiKZ diverges from this generalization in that its inner body is anchored on a vertex-proximal hexamer of the major capsid protein, not directly on the 12-fold portal. However in T4, which has two kinds of internal proteins in copious amounts (gpAlt and gpIPs), they are excluded from the peripheral zone that is occupied by shells of closely packed and highly ordered DNA but otherwise distributed seemingly at random through the capsid interior. Two papers reporting projects (2) and (3) are being prepared. 4) Hepatitis B virus is a major cause of acute and chronic liver disease. HBV is a small, enveloped, DNA virus whose core gene codes for two variants: core antigen (cAg), which assembles into capsids; and a precore protein, which is secreted as the non-particulate e-antigen (eAg). The cAg subunit consists of an assembly domain (residues 1-149) and a C-terminal domain (residues 150-183). eAg is directed to the ER by a 29-residue signal peptide. Mature eAg consists of the assembly domain plus 10 residues of the signal peptide. Previously we showed that the propeptide causes a radically altered mode of dimerization for eAg relative to cAg. In FY17 we performed structural studies in which a scFv was used as a crystallization chaperone, and obtained a co-crystal that diffracted to 0.173 nm. When compared with our earlier structure solved at 0.320 nm, the new structure shows distinct conformational differences between the paired eAg monomers, indicating a flexibility that may have functional implications. Our earlier work on this system included cryo-EM localizations of key epitopes on the capsid surface. We have now turned our attention to crystallographic studies of eAg complexed with fragments of monoclonal antibodies. A paper describing these structures is in preparation. We also investigated the immunological profile of eAg, producing a panel of chimeric rabbit/human monoclonal Fabs selected by phage display. These Fabs were expressed in E. coli, purified and characterized. Some had unprecedentedly high binding affinities and high specificity. The Fabs were used to develop a new and quantitative ELISA-based assay for eAg that was found to be superior in specificity and sensitivity to existing commercial assays. We also purified eAg from individual patient plasmas by microscale immunoaffinity chromatography. Western blotting and mass spectrometry indicated essentially structural identity with recombinant eAg apart from minor C-terminal heterogeneities. A paper has been submitted for publication.